The recent explosion of information in the fields of genomics and proteomics has provided a rich ground for the discovery of molecular targets against which therapeutic and/or diagnostic agents can be directed. Tissues for potential target discovery may include tumors and other malignant growths, or infected or inflamed tissues. For example, methods have been described for gene expression profiling of tumor cells (see any one of Ono et al. (2000) Cancer Res. 60(18):5007-11; Svaren et al. (2000) J Biol Chem.; or Forozan et al. (2000) Cancer Res. 60(16):4519-25 for examples). Similarly, proteomics has been used to profile the protein expression in tumor samples (see Minowa et al. (2000) Electrophoresis 21(9):1782-6; Cole et al. (2000) Electrophoresis 21(9):1772-81; Simpson et al. (2000) Electrophoresis 21(9):1707-32); etc.
Cancer is caused by multiple genetic events that result in the activation of proto-oncogenes and/or the inactivation of tumor suppressor genes. In some areas of the world, cancer has become or shortly will become the leading disease-related cause of death of the human population. For example, in the United States, cancer is the second leading cause of death behind cardiovascular disease, and it is projected that cancer will become the leading cause of death within a few years. The medical treatment of cancer still has many unmet needs. Surgery and radiation are generally only successful if the cancer is found at an early, localized stage. Once the disease has progressed to locally advanced cancer or metastatic disease, these therapies are less successful. Existing chemotherapeutic treatments are largely palliative in these advanced tumors, particularly in the case of the common epithelial tumors such as lung, colorectal, breast, prostate, and pancreatic cancers. Although a few chemotherapeutic regimens have yielded lasting remissions or cures (for example, in testicular cancer and childhood leukemias), it is clear that new therapeutic options are necessary.
The transformation and malignant growth of tumor cells is a complex process, which can be variable even within a particular tissue type. Analytical methods that can define the phenotype of tumor cells are useful in determining appropriate therapy, and are therefore of clinical interest. Additionally, knowledge of the mechanism by which a chemotherapeutic agent acts is useful determining optimal formulation and dosage of such agents; in screening for agents effective in treating cancer; and in following patients through a course of treatment.
A variety of post translational modifications of proteins take place. These modifications include phosphorylation, glycosylation, prenylation, and the like. The modifications, particularly reversible phosphorylation, can be a molecular mechanism by which intracellular signals are transmitted. A substantial number of signaling proteins are kinases or phosphatases that act on serine, threonine, and tyrosine residues. Wth over 2000 human genes predicted to code for kinases and the potential for each kinase to act on multiple targets, signaling networks are immensely complex. An important step towards unraveling this complexity is the development of new proteomics technologies that can quantitatively monitor the phosphorylation states of signaling proteins in a multiplex fashion. Such technologies would enable the detailed analysis of signaling pathways in a global perspective and the rapid identification of previously unrecognized signaling events.
Phosphorylation of target proteins by kinases is an important mechanism in signal transduction and for regulating enzyme activity. Tyrosine kinases (TK) are a class of over 100 distinct enzymes that transfer a phosphate group from ATP to a tyrosine residue in a polypeptide (Table 1). Tyrosine kinases phosphorylate signaling, adaptor, enzyme and other polypeptides, causing such polypeptides to transmit signals to activate (or inactive) specific cellular functions and responses. There are two major subtypes of tyrosine kinases, receptor tyrosine kinases and cytoplasmic/non-receptor tyrosine kinases.
To date there have been approximately 60 receptor tyrosine kinases (RTKs; also known as tyrosine receptor kinases (TRK)) described in humans. These kinases are high affinity receptors for hormones, growth factors and cytokines (Robinson et al. (2001) Oncogene 19:5548-57). The binding of hormones, growth factors and/or cytokines generally activates these kinases to promote cell growth and division. Exemplary kinases include insulin-like growth factor receptor, epidermal growth factor receptor, platelet-derived growth factor receptor, etc. Most receptor tyrosine kinases are single subunit receptors but some, for example the insulin receptor, are multimeric complexes. Each monomer contains an extracellular N-terminal region, a single transmembrane spanning domain of 25-38 amino acids, and a C-terminal intracellular domain. The extracellular N-terminal region is composed of a very large protein domain which binds to extracellular ligands e.g. a particular growth factor or hormone. The C-terminal intracellular region provides the kinase activity of these receptors. Receptor tyrosine kinases are key regulators of normal cellular processes and play a critical role in the development and progression of many types of cancer (Zwick et al. (2001) Endocr. Relat. Cancer 8:161-173).
Cancer is frequently associated with the abnormal expression and phosphorylation of oncogenes. Specific tumors may be characterized by the discrete activation of specific oncogenes such as MYC, BCL2 (encoding B cell lymphoma protein-2) and BCR-ABL. Targeted inactivation of oncoproteins is emerging as a specific and effective therapy for cancer. The best known example of a targeted therapy is imatinib mesylate, a small molecule that inactivates several tyrosine kinases, including the BCR-ABL tyrosine kinase in CML. Imatinib treatment results in tumor cell signaling changes in vitro, leading to cell death. In general, the ability to detect specific oncoproteins and their activation state is likely to be highly useful toward the development of new therapeutics as well as in monitoring the effectiveness of these treatments and in evaluating apparent therapeutic resistance13,21.
Current methods of protein detection are insensitive to detecting subtle changes in oncoprotein activation that underlie key cancer signaling processes. The requirement for large numbers of cells precludes serial tumor sampling for assessing a response to therapeutics. The present invention addresses this need.